鬼臼毒素及其衍生物

鬼臼毒素作为鬼臼属植物的主要活性成分,已经有广泛的研究报道。其同系物及衍生物具有很强的抗肿瘤活性。依托泊甙、替尼泊甙已经成功地用于临床抗肿瘤治疗。优化结构、寻找新的鬼臼毒素类化合物资源替代品,已成为当前进一步研究的主要方向。     

Interest of lignans in prevention and treatment of cancers

Lignans are diphenolic compounds widely distributed in the plant kingdom. They are mainly localised in lignified tissues, seeds and roots. These molecules are involved in plant defence mechanisms, but are also interesting for human health. Flax lignans belonging to the phytoestrogens are metabolised after ingestion into enterolignans that may offer a protection against the onset and development of hormono-dependant cancers. In vitro studies based on mammalian cellular models tend to confirm their beneficial effects observed during epidemiological studies and give us insights about their mechanisms of action. The most studied lignan, podophyllotoxin, and its semi-synthetic derivatives (etoposide, teniposide, etoposide phosphate), are particularly interesting at a curative level due to their cytotoxic properties. These semi-synthetic derivatives are used in chemotherapy of lung cancer for example. However, the extensive use of these anticancer drugs will lead to the problem of podophyllotoxin supply. This molecule is currently extracted from the rhizomes and roots of an Indian species Podophyllum hexandrum which has subsequently become endangered. Strategies are investigated to obtain economically viable alternative sources of Podophyllotoxin from plants and in vitro cultures of several species. Among them, north american Podophyllum peltatum, Linum wild species, Hyptis, Anthriscus, Juniperus or Dysosma species which accumulate Podophyllotoxin or closely related derivatives, are good candidates. double dagger.     

Enhanced production of podophyllotoxin by Podophyllum hexandrum using in situ cell retention bioreactor

The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide. Batch cultivation of Podophyllum hexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively. The batch kinetics was used to identify the mathematical model. The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor. An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode.     

Detection and identification ofPodophyllotoxin produced by cell cultures derived from podophyllum hexandrum royle

The phenylpropanoid derived lignan podophyllotoxin, occurring inPodophyllum species, is used as a starting compound for the chemical synthesis of the antitumour agents etoposide (VP-16-213) and teniposide (VM-26). At present, the availability of this lignan becomes increasingly limited. As an alternative source, cell cultures originating fromPodophyllum hexandrum Royle were initiated. Analysis of the cell extracts using different HPLC systems as well as TLC, indicated the presence of podophyllotoxin. After prepurification of the extracts by means of ITLC, the identity was confirmed by mass spectrometric analysis. Dark-grown cultures accumulated considerable higher amounts of podophyllotoxin in comparison with the light-grown cultures.     

Tumour-inhibitory aryltetralin lignans from Podophyllum pleianthum

Roots of Podophyllum pleianthum contain eight aryltetralin lignans: podophyllotoxin, desoxypodophyllotoxin, podophyllotoxone, isopicropodophyllone and the four corresponding 4′-demethyl derivatives. The lignan pattern is very similar to that of P. hexandrum. A useful TLC spray reagent for Podophyllum lignans is described.     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

Biotechnological approaches for producing aryltetralin lignans from Linum species

The genus Linum includes more than 230 globally distributed species, which have attracted great interest as they grow rapidly and are already sources of commercially important products, e.g. flax and linseed oil. Furthermore, they contain lignans such as podophyllotoxin (PTOX), deoxypodophyllotoxin (a precursor of both PTOX and 6-methoxypodophyllotoxin, the latter via β-peltatin, and β-peltatin-A-methyl ether) and various derivatives. Lignans are natural compounds derived from two 8,8′-linked C₆C₃ (propylbenzene) units. PTOX is an aryltetralin-lignan with strong cytotoxic and antiviral activities. Thus, it is used as a starting material for producing various semisynthetic derivatives that are widely used in chemotherapy, such as etoposide, teniposide and etopophos. It is currently produced largely from Podophyllum hexandrum and P. peltatum, slow-growing endangered species of the Berberidaceae. Hence, the possibility of producing it from Linum, especially members of section Syllinum under either in vitro or ex vitro conditions is highly attractive. This review summarizes related research, focusing on in vitro production of aryltetralin lignans from various Linum species and possible biotechnological strategies to improve their production. The key pathways, enzymes and genes involved are highlighted and future challenges that must be met to allow viable, large-scale production of this anticancer drug lead are discussed.     

The production of cytotoxic lignans by plant cell cultures

Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.     

Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.     

Production of podophyllotoxin using cross-species coculture of Linum flavum hairy roots and Podophyllum hexandrum cell suspensions

Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.     

Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans

(-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 angstrom resolution, respectively. The enzyme is a homotetramer, consisting of an alpha/beta single domain monomer containing seven parallel beta-strands flanked by eight alpha-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD(+) and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.     

The American mayapple revisited—podophyllum peltatum—still a potential cash crop?

Podophyllum peltatum, was reexamined for its potential use in the commercial production of podophyllotoxin, a lignan used in the semisynthesis of important anticancer drugs. A survey of the natural population of the American mayapple, Podophyllum peltatum, was conducted in order to identify high-yielding genotypes. Plants were collected from the eastern and central United States. The lignan content of leaf blades and rhizome material of the collected specimens was characterized by aqueous extraction followed by HPLC analysis. Podophyllotoxin and α-peltatin appeared most prominently among the lignans obtained. Leaf blades were generally richer in podophyllotoxin than rhizomes. Several high-yielding accessions were identified, the blades of which contained 4.0–5.6% podophyllotoxin. A negative correlation was observed between podophyllotoxin and peltatin content in the blades. The combination of high biosynthetic capacity and preferential accumulation of podophyllotoxin in leaves of mayapple makesthis plant an excellent candidate for agricultural production of podophyllotoxin.     

Biotechnological interventions for harnessing podophyllotoxin from plant and fungal species: current status,challenges, and opportunities for its commercialization

Podophyllotoxin is an aryltetralin lignan synthesized in several plant species, which is used in chemotherapies for cancers and tumor treatment. More potent semisynthetic derivatives of podophyllotoxin such as etoposide and teniposide are being developed and evaluated for their efficacy. To meet the ever increasing pharmaceutical needs, species having podophyllotoxin are uprooted extensively leading to the endangered status of selective species mainly Sinopodophyllum hexandrum. This has necessitated bioprospection of podophyllotoxin from different plant species to escalate the strain on this endangered species. The conventional and non-conventional mode of propagation and bioprospection with the integration of biotechnological interventions could contribute to sustainable supply of podophyllotoxin from the available plant resources. This review article is focused on the understanding of different means of propagation, development of genomic information, and its implications for elucidating podophyllotoxin biosynthesis and metabolic engineering of pathways. In addition, various strategies for sustainable production of this valuable metabolite are also discussed, besides a critical evaluation of future challenges and opportunities for the commercialization of podophyllotoxin.     

Biosynthesis of Podophyllum lignans—II. Interconversions of aryltetralin lignans in Podophyllum hexandrum

Feeding experiments in Podophyllum hexandrum plants with labelled aryltetralin lignans have established much of the biosynthetic interrelationships existing amongst Podophyllum lignans. Thus, desoxypodophyllotoxin is converted into podophyllotoxin, which in turn is oxidized to podophyllotoxone, although this latter step appears to be reversible. A similar sequence is proposed for the corresponding 4′-demethyl derivatives. Although 4′-demethyldesoxy-podophyllotoxin is readily converted into 4′-demethylpodophyllotoxin, neither compound is incorporated into lignans of the 4′-methyl series such as podophyllotoxin. The Podophyllum lignans may be subdivided biogenctically into two groups, those with 3,4,5-trimethoxy substitution in the pendent aryl ring, and those with a 4-hydroxy-3,5-dimethoxy substituted pendent ring, although these probably arise from a common precursor. A biogenetic scheme interrelating all of the known Podophyllum aryltetralin lignans is proposed.     

Synthesis and biological evaluation of novel 4β-(1,3,4-oxadiazole-2-amino)-podophyllotoxin derivatives

A series of new 4β-(1,3,4-oxadiazole-2-amino)-podophyllotoxin derivatives were designed and synthesized. Their cytotoxicity in vitro against six tumor cell lines (DU-145, SGC-7901, A549, SH-SY5Y, HepG2 and HeLa) were evaluated by standard MTT assay. The pharmacological results showed that most of the newly synthesized podophyllotoxin derivatives displayed potent cytotoxicity against at least one of the tested tumor cells; and among the new derivatives, 11b was more potent than podophyllotoxin against HepG2 and Hela cell lines. Furthermore, 11b exhibited much better selectivity toward the normal cell lines L929 and Vero than etoposide, 5-Fu and podophyllotoxin. The possible antitumor mechanism of 11b is to inhibit the activity of DNA topoisomerase II, result in the S-phase arrest, and then cause apoptotic cell death.     

Identification,validation, and expression of ABC transporters in Podophyllum hexandrum and their role in podophyllotoxin biosynthesis

Podophyllum hexandrum Royle is an important medicinal herb of North-Western Himalayas, and podophyllotoxin, being its major metabolite, has been used extensively in the preparation of several anticancer drugs. Podophyllotoxin accumulates in rhizomes; however, no information exists on the role of ATP-binding cassette (ABC) transporters vis-à-vis podophyllotoxin content. The present study reports identification, validation, and expression analysis of ABC transporter genes from P. hexandrum. Total 252 ABC transporter genes were identified as unigenes out of which 22 were further validated using real time qPCR in different tissues of varying podophyllotoxin content. Differential expression analysis and Pearson’s correlation coefficient revealed two candidate genes PhABC6 and PhABCIII having a positive correlation with the podophyllotoxin content. PhABCIV showed the highest expression in rhizomes (20.53-folds compared to shoots) suggesting its possible role in transport and accumulation of podophyllotoxin.     

On the improvement of the podophyllotoxin production by phenylpropanoid precursor feeding to cell cultures of Podophyllum hexandrum Royle

In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferin into podophyllotoxin, maximally a 12.8 fold increase in content was found. Permeabilization using isopropanol, in combination with coniferin as a substrate, did not result in an extra increase in podophyllotoxin accumulation. Concentrations of isopropanol exceeding 0.5% (v/v) were found to be rather toxic for suspension growth cells of P. hexandrum. When coniferin was fed in presence of such isopropanol concentrations, -glucosidase activity was still present, resulting in the formation of the aglucon coniferyl alcohol. In addition, podophyllotoxin was released into the medium under these permeabilization conditions. Entrapment of P. hexandrum cells in calcium-alginate as such or in combination with the feeding of biosynthetic precursors, did not improve the podophyllotoxin production. Cell-free medium from suspension cultures at later growth stages incubated with coniferin, resulted in the synthesis of the lignan pinoresinol.     

Design,synthesis and cytotoxic activity of novel sulfonylurea derivatives of podophyllotoxin

Three series of novel sulfonylurea podophyllotoxin derivatives were designed, synthesized, and evaluated for in vitro cytotoxicity against four tumor cell lines (A-549, DU-145, KB and KBvin). Compounds 14c (IC₅₀: 1.41–1.76 μM) and 14e (IC₅₀: 1.72–2.01 μM) showed superior cytotoxic activity compared with etoposide (IC₅₀: 2.03 to >20 μM), a clinically available anticancer drug. Significantly, most of the compounds exhibited comparable cytotoxicity against the drug-resistant tumor cell line KBvin, while etoposide lost activity completely. Preliminary structure–activity relationship (SAR) correlations indicated that the 4′-O-methyl functionality in podophyllotoxin analogues may be essential to maintain cytotoxic activity, while an arylsulfonylurea side chain at podophyllotoxin’s 4β position can significantly improve cytotoxic activity.